Chromosome banding and genome compartmentalization in fishes

The diploid chromosome number of the Chinese raccoon dog varies from 54 (no B chromosomes) to 58 (4 B chromosomes). The B chromosomes are totally heterochromatic. An electron microscopic study was made of the synaptonemal complexes (SC) in spermatocytes of these animals. The SC karyotype consists of 27 regular chromosome pairs (autosomes and the sex chromosomes) plus the B chromosomes. The Bs pair effectively with one another at pachytene, but the SC axes of the B chromosomes are much denser than those of the A chromosomes. Depending on the number of Bs, both bivalents and multivalents have been observed. When three B chromosomes are present in a cell, parallel alignment of all three SCs can be seen. Formation of multivalents indicates high homology among these supernumerary heterochromatic chromosomes. Fusiform bulges are found along unpaired regions of all chromosomes which are particularly pronounced in diplotene.     

Variation of B Chromosome Associated with Tissue Culture in Wheat-rye Cross

In vitro variation of B chromosomes was studied by examining the callus cells derived from the immature embryos from a cross of Chinese Spring wheat ( Triticum aestivum L.) and Fin 7416 rye ( Secale cereale L.) carrying two B chromosomes. In 40-d-old callus cells, the numbers of B chromosomes ranged from one to four in 65.6% of the cells observed. The distribution of B chromosome numbers was associated with the ploidy levels of the normal chromosomes (A chromosomes). The frequency of the cells with high numbers of B chromosomes (i.e., three or four B chromosomes) in the amphiploid cells with 56 A chromosomes was greater than those in the haploid cells with 28 A chromosomes. Although structural changes in the rye A chromosomes were observed, cytological observation and genomic in situ hybridization demonstrated that the rye B chromosomes were conserved in morphological appearance following tissue culture.     

丽纹攀蜥粗线期染色体核型及微小染色体的分析

本文以精巢、四肢骨和脊椎骨为材料,利用直接制片法制备了丽纹攀蜥（Japalura splendida）染色体标本,首次对其精巢减数分裂粗线期染色体,特别是微小染色体的长度进行了测量,描述了染色体特征,并进行了核型分析.同时与二倍体细胞染色体的核型进行了比较,发现11对微小染色体类型中包括8对中着丝粒染色体、2对端着丝粒染色体和1对点状染色体.     

Nonreplication of heterochromatic chromosomes in a mealy bug,Planococcus citri (Coccoidea: Homoptera)

In males of mealy bugs with the lecanoid chromosome system, the paternal set of chromosomes becomes heterochromatic in early embryogeny. In males of the mealy bug, Planococcus citri, the heterochromatic (H) set in testis sheath cells and in most of the oenocytes apparently did not replicate while the euchromatic (E) set was undergoing several cycles of endoreplication. In third instar males, testis sheath cells in endoanaphase and endotelophase exhibited 5H and either 40 or 80E chromosomes. The increase in the number of E chromosomes was attributed to the replication of only the E chromosomes. Oenocytes of third instar males had 0, 5, or 10H chromosomes and from 10 to 240E chromosomes. The oenocytes with 5H chromosomes had a mean of 50.8E chromosomes, and those with 10H chromosomes had a mean of 155.6E chromosomes. Nuclear and cell fusion was considered as a means of producing the various numbers of H and E chromosomes in oenocytes, and it was concluded that although nuclear fusion probably took place, the differences between the number of H and E chromosomes was at least in part due to replication of only the E chromosomes. The size of the H chromosomes was about the same in all the testis sheath cells and the oenocytes irrespective of the level of endopolyploidy for the E set. These H chromosomes apparently did not increase in polyteny, because they were only about half the size of the H chromosomes in prophase I of spermatogenesis. The significance of the nonreplication of the H set and the control of nonreplication are briefly discussed.This study was aided by a grant (GB-1585) from the National Science Foundation, Washington, D.C.     

Biological effects of a bifunctional DNA crosslinker. I. Generation of triradial and quadriradial chromosomes.

Interduplex crosslinks by a bifunctional anthramycin DNA crosslinker produced triradial and quadriradial chromosomes. The crosslinker alkylates guanine at N-2. Bovine chromosomes contain GC-rich density satellite DNAs at the centromeric heterochromatin and is the basis for the formation of triradial and quadriradial chromosomes at the centromeres. The in situ crosslinking of interphase chromosomes indicates that the distance between centromeres is 17.5 A. We conclude that the nuclear matrix associated DNA in the centromeric heterochromatin of interphase chromosomes are positioned close enough for crosslinking to occur. We propose a model for the generation of triradial and quadriradial chromosomes based upon the number of interduplex crosslinks between two chromosomes.     

Zebrafish chromosome banding.

Banding techniques were carried out on metaphase chromosomes of zebrafish (Danio rerio) embryos. The karyotypes with the longest chromosomes consist of 12 metacentrics, 26 submetacentrics, and 12 subtelocentrics (2n = 50). All centromeres are C-band positive. Eight chromosomes have a pericentric C-band in each arm and 22 chromosomes have one in the longest arm. Two chromosomes have a slightly heterochromatic long arm and five chromosomes have an Ag-NOR at the terminal end of the long arm. Other banding patterns and sex chromosomes could not be revealed.     

The composition and structure of isolated chromosomes

1. The preparation of isolated chromosomes from liver, kidney, and pancreas has been described. 2. It has been shown that there is no gross cytoplasmic contamination in these preparations. 3. In a microscopic study of isolated chromosomes the same chromosomes have been found in different tissues of the same organism. Since individuality is one of the main characteristics of chromosomes, there can be little doubt that the preparations do, in fact, contain isolated chromosomes. 4. A quantitative study of staining with crystal violet shows that this basic dye competes with histone for the phosphoric acid groups of the DNA in chromosomes. The displacement of histone by protamine has been demonstrated. 5. Preparation of histone-free chromosomes has been described. Removal of histone does not affect the microscopic appearance of chromosomes. 6. The non-histone or residual protein has been prepared from histone-free chromosomes. The quantity of residual protein in a preparation of chromosomes is correlated with the amount of cytoplasm in the cells from which the chromosomes were prepared. 7. The microscopic appearance of chromosomes depends upon the association of DNA with residual protein. 8. Evidence has been given that in a chromosome there are two DNA-containing nucleoproteins; in one DNA is combined with histone, and in the other it is combined with residual protein.     

DNA image-fluorimetry of individual human chromosomes

A microfluorimetric method has been developed for determination of DNA content in individual human chromosomes. The method is based on a preliminary identification of chromosomes with Hoechst 33258 followed by staining of the chromosomes with Feulgen reaction by using Schiff’s reagent type ethidium bromide-SO₂ and then by measuring the fluorescence intensity of the chromosomes by using an image analyzer. The method allows determining the DNA content of individual chromosomes with an accuracy up to 4.5 fg. The DNA content of individual human chromosomes and their p-and q-arms, as well as homologous chromosomes, were measured by using the developed method. It has been shown that the DNA content in chromosomes of the normal human karyotype is unstable and can fluctuate in some chromosomes within 35–40 fg.     

Chromosome analysis and sorting in <Emphasis Type="Italic">Vicia sativa</Emphasis> using flow cytometry

Procedures were developed for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) of common vetch (Vicia sativa L., 2n=12). Suspensions of intact chromosomes were prepared from root tips after cell cycle synchronization, formaldehyde fixation, and mechanical homogenization. On average, 3 × 10⁵ morphologically intact chromosomes could be isolated from 25 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing four peaks, representing particular chromosomes and/or pairs of chromosomes with similar relative DNA content. Peaks I and II were assigned to chromosomes 6 and 5, respectively. These chromosomes could be sorted with a purity exceeding 90 %. The two remaining peaks on the flow karyotype were composite, each of them representing a pair of chromosomes. Chromosomes 1 and 3 were assigned to composite peak III while chromosomes 2 and 4 were assigned to composite peak IV. The chromosomes could be sorted with a purity of 99 % from both composite peaks. Bivariate flow karyotyping after simultaneous staining of chromosomes with DAPI and mithramycin was not found helpful in discriminating additional chromosomes. This study extends the number of legume species for which flow cytogenetics is available and provides a new tool for targeted and effective analysis and mapping of common vetch genome.     

Two supernumerary marker chromosomes, originating from chromosomes 6 and 11, in a child with developmental delay and craniofacial dysmorphism

The interpretation of the significance of marker chromosomes, which can be encountered at prenatal diagnosis, is extremely problematic. Various factors contribute to the difficulty of clarifying the phenotypic risks of supernumerary marker chromosomes, including differences in the size, structure, and origin of marker chromosomes, as well as the occurrence of multiple marker chromosomes of different origin in the same proband. Research on marker chromosomes is currently in a data-accumulation phase. We report the presence of two marker chromosomes, originating from chromosomes 6 and 11, in a child with developmental delay and craniofacial dysmorphism and discuss the related literature.     

Discrimination between groups of chromosomes and individual chromosomes in the normal human karyotype

The karyotypes of 200 normal individuals are examined in a study designed to investigate the extent to which the cytologist's classification of chromosomes into the A–G autosomal groups and identification of individual chromosomes are reflected in the values of measurements made on these chromosomes. The statistical technique of discriminant function analysis is employed to obtain linear functions of the chromosomal measurements which may be used to classify and identify chromosomes. The results of the study indicate that while chromosomes can be grouped with a reasonably high probability of success using measurements alone, identification of individual chromosomes is somewhat more difficult.     

Mapping of KIT adjacent sequences on canid autosomes and B chromosomes

B chromosomes are often considered to be one of the most mysterious elements of karyotypes (Camacho, 2004). It is generally believed that mammalian B chromosomes do not contain any protein coding genes. The discovery of a conserved KIT gene in Canidae B chromosomes has changed this view. Here we performed analysis of sequences surrounding KIT in B chromosomes of the fox and raccoon dog. The presence of the RPL23A pseudogene was shown in canid B chromosomes. The 3' end fragment of the KDR gene was found in raccoon dog B chromosomes. The size of the B-specific fragment homologous to the autosome fragment was estimated to be a minimum of 480 kbp in both species. The origin and evolution of B chromosomes in Canidae are discussed.     

STUDIES ON THE ISOLATION OF METAPHASE CHROMOSOMES

A method for the isolation of metaphase chromosomes from mouse L1210 leukemia cells has been developed. Cells, arrested at metaphase with colchicine, were exposed to hypotonic solution and the pH was then adjusted to 5.6 to stabilize the chromosomes. The metaphase figures were subsequently disrupted and the chromosomes isolated by a series of differential centrifugations in sucrose. The isolated chromosomes were well preserved, as judged by morphological criteria. The effect of various enzymes and chemical agents on the isolated chromosomes was studied. Chymotrypsin, trypsin, and deoxyribonuclease caused a marked disintegration of the chromosomes, whereas treatment with pepsin and ribonuclease induced no significant morphological alterations.     

Bivariate flow cytometry of farm animal chromosomes: a potential tool for gene mapping

Bivariate flow karyotypes of chromosomes from sheep, cattle and pig lymphocytes and from a cattle-mouse somatic cell hybrid line were obtained using a dual laser fluorescence-activated cell sorter (FACS). Pig chromosomes were resolved into 19-20 peaks, indicating that most, if not all, pig chromosomes could be separated by this technique. Sheep chromosomes showed incomplete separation but three clear peaks, presumably representing the three large metacentric chromosomes, plus five other clusters were obtained. Cattle chromosomes showed poor separation but about four peaks could be distinguished, indicating that certain chromosomes could be sorted in this species. The use of cattle-mouse hybrids may enable other individual cattle chromosomes to be obtained. It is concluded that FACS separation will be a useful additional tool for gene mapping.     

Centromere organization and UU/V sex chromosome behavior in a liverwort

In 1917, sex chromosomes in plants were discovered in a liverwort with hetermorphic U and V chromosomes. Such heteromorphy is unexpected because, unlike the XY chromosomes in diploid-dominant plants, in haploid-dominant plants the female U and the male V chromosomes experience largely symmetrical potential recombination environments. Here we use molecular cytogenetics and super-resolution microscopy to study Frullania dilatata, a liverwort with one male and two female sex chromosomes. We applied a pipeline to Illumina sequences to detect abundant types of repetitive DNA and developed FISH probes to microscopically distinguish the sex chromosomes. We also determined the phenotypic population sex ratio because biased ratios have been reported from other liverworts with heteromorphic sex chromosomes. Populations had male-biased sex ratios. The sex chromosomes are monocentric, and of 14 probes studied (eight satellites, five transposable elements and one plastid region), four resulted in unique signals that differentiated the sex chromosomes from the autosomes and from each other. One FISH probe selectively marked the centromeres of both U chromosomes, so we could prove that during meiosis each U chromosome associates with one of the opposite telomeres of the V chromosome, resulting in a head-to-head trivalent. The similarity of the two U chromosomes to each other in size and in their centromere FISH signal positions points to their origin via a non-disjunction event (aneuploidy), which would fit with the general picture of sex chromosomes rarely crossing-over and being prone to suffer from non-disjunction.     

两栖动物性染色体的多样性及其进化机制的研究进展

两栖动物性别决定类型和性染色体具有多样性的特点。在已发现异形性染色体两栖动物中,大部分物种Y或W染色体大于其对应的X或Z染色体,少数物种具有高度分化的Y或W染色体。同时两栖动物类群内基因组大小差异大,性染色体间分子水平上也存在差异。高频转换、偶然重组和染色体重排可能是两栖动物性染色体进化过程中的关键机制。本综述通过对两栖动物性染色体进化的深入探讨,揭示其遗传性别决定的机理,有助于对两栖动物性别人工调控的进一步探索。     

The cytology of oxalis dispar (brown)

Summary The chromosome complement ofOxalis dispar 2n = 12 consists of a pair of submedian (SM) chromosomes, three subterminal (ST) and seven telocentric (TE) chromosomes. At mitosis and meiosis the TE chromosomes are as efficient and stable as the other chromosomes of the complement. Chromosome breakage at the centromere in the metacentric chromosomes may give rise to new TE chromosomes. It is suggested that all the TE chromosomes may have arisen from metacentric chromosomes in this way. They may have been favoured because of the deleterious nature of the discarded chromosome arms.     

B chromosomes have a functional effect on female sex determination in Lake Victoria cichlid fishes

The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.     

Homologous chromosomes characteristics by sequential banding procedures in rainbow trout Oncorhynchus mykiss

The development of high resolution methods of chromosome banding helped the finding of homologous chromosomes, detecting chromosomal abnormalities, and assigning the gene loci to particular chromosomes in mammals. Unfortunately, small and numerous fish chromosomes do not show GC rich and GC poor compartments, this preventing the establishment of G banding pattern. The combination of techniques enabling the identification of constitutive heterochromatin (C-banding), heterochromatin resistant to restriction endonucleas, NOR bearing chromosomes (AgNO3 banding), or AT rich regions on chromosomes (DAPI banding) in sequential staining provides a better characteristic of fish chromosomes. In this work sequentially DAPI, DdeI, AgNO3 stained chromosomes of rainbow trout resulted in the characteristic banding pattern of some homologous chromosomes. Procedure of FISH with telomere probe and DAPI as a counterstaining fluorochrome visualized simultaneous hybridization signals and DAPI banding. Possibility of detection both FISH and DAPI signals can help in procedures of gene mapping on chromosomes.